Why certain proteins are unable to express the spatial information encoded in their amino acid sequences without the aid of molecular chaperones is not fully understood. Yet protein misfolding underlies devastating pathologies, such as cystic fibrosis, thalassemias, and a variety of amyloid neuropathies such as Alzheimer's and Huntington's disease. The long-term goal of this proposal is to understand how molecular chaperones guide proteins to their final, active three dimensional structures. Toward this end, we intend to focus on one subclass of the molecular chaperones, the ubiquitous, ring-shaped complexes known as chaperonins. One member of this conserved and essential family of proteins is the bacterial GroEL-GroES complex. Using the GroEL-GroES system as a model, along with model folding substrates, we propose experiments intended to uncover general principles for chaperone-dependent protein folding. In order to study the dynamics of the GroEL chaperonin and how it interacts with protein folding intermediates, we are developing fluorescence and rapid mixing methods. Previously, we successfully used fluorescence energy transfer (FRET) to follow the sequence of steps that drive the GroEL reaction cycle. This method relies on the introduction of cysteine residues into GroEL, GroES and substrate protein, which are then labeled with fluorescent probes. We now extend this approach to include time-resolved FRET measurements, in order to systematically map the morphology of a GroEL-bound folding intermediate. We anticipate that this combined approach will allow us to determine why the GroEL-GroES chaperonin is required to fold certain proteins and how specific interactions between these proteins, GroEL, and GroES facilitate productive folding. Our aims are: (1) to develop a FRET assay which can be used to map the morphology of a GroEL bound folding intermediate, (2) to apply this assay to follow structural changes in a folding intermediate while bound to GroEL in order to test two models of GroEL-stimulated folding and (3) to determine how GroEL-dependent protein folding is triggered, by testing specific models of substrate encapsulation beneath GroES.